1. Field of the Invention
The present invention is directed to improvements in screening methods used to detect, amplify and select microorganisms subjected to mutagenesis which thereby develop desirable characteristics differing from the microorganisms prior to mutagenesis. More particularly, the present invention is directed to novel screening methods which enhance the growth of certain desired mutants in a pool of organisms which has been subjected to random mutagenesis in such a manner to permit screening of a large number of potential mutants efficiently and effectively.
2. State of the Art
In the prior art, several methods of directing the evolution of a pool of microorganisms subjected to random mutagenesis have been developed. One manner of effecting focused evolution of an organism is to introduce novel characteristics into protein products by site-directed mutagenesis. However, such methods require a high level of knowledge regarding the subject protein in terms of structure and function relationships and the specific effects desired. As a result, methods have been developed which take advantage of random mutational events over a large cell population and/or over several generations to produce a small number of desired mutations. Such methods, generally referred to as directed evolution, require the steps of obtaining a pool of starting material microorganisms, subjecting these microorganisms to random mutagenesis and subsequently applying appropriate selection techniques to select mutants having desired characteristics.
Essential to the success of directed evolution techniques are screening or selection techniques for isolating desired mutants. A common selection technique involves growing the mutant organisms under conditions such that only desired mutants exhibit growth. One such technique is to plate the mutant organisms on media which limits growth to microorganisms having a specific (mutant) characteristic. For example, media which includes a toxin will force selection of organisms having resistance to the toxin. Similarly, media which includes a substrate for which it is necessary that the mutant organisms be able to metabolize that substrate for survival will limit growth to those organisms capable of producing the appropriate enzymes. Further, the mutant organisms can be subjected to rigorous conditions to determine if, for example, the mutant organisms are better suited for surviving at high temperature or pH.
An example of such selection pressure is illustrated in Forney et al., Appl. Environ. Microb., vol. 55, no. 10, pp. 2550-2555 (1989), wherein mutant penicillin amidases with novel substrate specificity were obtained by mutagenizing a strain of E.coli and selecting on the ability of the mutants to hydrolyze glutaryl-(L)-leucine and provide leucine to Leu.sup.- strains. Cells which retained the wild type enzyme would not grow on the minimal media containing glutaryl-(L)-leucine as the sole source of leucine. The authors reported that the growth rates of the Leu.sup.- cells that expressed mutant amidases increased as the glutaryl-(L)-leucine concentration increased or as the medium pH decreased. As a result, it was possible for the authors to deliberately modify the substrate specificity of penicillin amidase and select mutants with amidases that were progressively more efficient at hydrolyzing glutaryl-(L)-leucine.
An alternative technique which was developed to screen mutant proteins is known as phage display. This technique is valuable in isolating randomly mutagenized DNA encoding proteins having a specific desired binding activity. In the phage display technique, a lambda phage or equivalent is produced which contains a mutagenized gene encoding a specific binding protein. The phage is then subjected to a ligand binding procedure, e.g., column affinity purification, wherein the ligand is selected based on a desired binding capability of the mutant binding protein and can thus selectively bind to and trap phage displaying the desired mutant binding protein on its surface. Subsequent to isolation, the trapped phage can be used to infect appropriate bacterial hosts to produce larger quantities of protein. One example of phage display is application as a tool for isolating proteases having new protease specificities, Corey et al., Gene, vol. 128, pp. 129-134 (1993), wherein phage display was utilized to identify mutant trypsin molecules which comprised a fusion product of the trypsin molecule and M13 coat proteins through capture by immobilized ecotin as a ligand.
The above methods have provided powerful mechanisms for applying selection pressure to effect isolation of specific desired mutants among a randomly mutagenized population. However, a problem remains in screening large numbers of organisms in an efficient manner. For example, plate screening requires serial dilutions of liquid cultures to the extent that only a small number of colonies exist on each plate. Where the mutant pool comprises millions of organisms, this imposes an enormous procedural burden on the researcher to analyze the entire population.
This problem is amplified where less stringent selection criteria are used to detect certain types of mutations, for example, improved catalytic activity, temperature or pH resistance in an enzyme. One example of such a selection technique involves use of media which includes a necessary cellular nutrient, access to which requires a specific mutation in an enzyme. In such instances, it is often advantageous to repeatedly mutate a population of microorganisms, screening between each mutational event to select for incrementally improved mutants. Because the mutants will have varying degrees of viability on the media as opposed to absolute viability or not, it becomes necessary to screen a much larger quantity of organisms thus adding to the procedural burden on the researcher.
Moreover, if the selection procedure involves the production of a necessary nutrient by a mutant extracellular enzyme, organisms lacking the necessary mutation will benefit from the production of mutant enzymes by other colonies due to nutrient diffusion through the media. Thus, it is necessary to drastically limit the number of colonies on a media plate through serial dilutions, while at the same time observing a large increase in the number of mutants analyzed. The combination of these effects means that on the order of 50-100 colonies per plate becomes a maximum. Due to these effects, plus the additive effect of mutagenizing over several generations, the burden becomes prohibitive if entire populations of microorganisms are to be screened. To avoid such burdensome screening, many researchers opt to analyze only a minor segment of the entire population, thus reducing the potential for detecting desired mutants.
From the above, it is apparent that advances have been made in the development of screening techniques for accurately identifying and isolating desired characteristics in proteins produced from randomly mutagenized genes. However, a problem remains in the art related to the large scale screening of mutants, particularly in the field of improved mutant of extracellularly produced enzymes. Given the miniscule proportion of desired mutant organisms produced during random mutagenesis in comparison with the total pool of microorganisms, it would be advantageous to find an accurate technique for the large scale screening and isolation of mutant organisms subsequent to random mutagenesis. Thus, a system which allows simple, speedy, accurate and efficient isolation of mutant organisms from a mutagenized pool on the order of 10,000 to 1,000,000 organisms without crossover of nutrients would greatly improve present techniques. The prior art, however, fails to provide such a system.